High affinity human antibodies to human il-18 receptor

ABSTRACT

An isolated antibody or antibody fragment that binds human interleukin-18 receptor alpha (hIL-18Rα), comprising a light chain variable region (LCVR) selected from the group consisting of SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 61, 65, 69, 73, 77, and 81 and/or a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, and 79, or a fragment or sequence modified by an amino acid substitution, deletion or addition thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.11/784,308, filed Apr. 6, 2007, which claims the benefit under 35 USC§119(e) of U.S. Provisional application Ser. No. 60/790,081, filed Apr.7, 2006, which applications are herein specifically incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention related to human antibodies and antibody fragmentsspecific for human interleukin 18 receptor alpha (hIL-18Rα) (SEQ IDNO:1), pharmaceutical compositions, and therapeutic methods thereof.

SEQUENCE LISTING

An ASCII compliant text file of the sequence listing is filedconcurrently with the present specification (37 CFR §1.52(e) and 37 CFR§1.821). The contents of the text file are herein incorporated byreference. The text file containing the sequence listing is named “6000BSEQ LIST”, was created on Oct. 9, 2009, and contains 84,560 bytes.

STATEMENT OF RELATED ART

Two interleukin-18 receptors are known, IL-18Rα and IL-18Rβ. IL-18Rα isalso known as 2F1, interleukin-1 receptor related protein 1 (IL-1 Rrp1),IL-18R1, IL-1R5, IL-1 Rrp and IL-1 receptor-like (Parnet et al. U.S.Pat. No. 5,776,731; U.S. Pat. No. 6,090,918; Sims et al. U.S. Pat. No.6,589,764. U.S. Pat. No. 6,600,022 (Torigoe et al.) discloses a murinemonoclonal antibody specific to IL-18Rα comprising variable heavy andlight chain protein sequences.

Methods to produce antibodies useful as human therapeutics includegeneration of chimeric antibodies and humanized antibodies (see, forexample, U.S. Pat. No. 6,949,245). WO 94/02602 (Abgenix) (hereinspecifically incorporated by reference) describes a method of generatingnonhuman transgenic mice capable of producing human antibodies.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides an agent, preferably arecombinant human antibody, that specifically bind human interleukin-18receptor alpha (hIL-18Rα) (SEQ ID NO:1). The binding agents of theinvention are characterized by binding to hIL-18Rα with high affinityand by the ability to neutralize hIL-18 activity, includingIL-18-induced interferon gamma expression and IL-18-induced cellularactivation. The agents can be antibodies that are full-length (forexample, an IgG1, IgG2, IgG4, IgG4 mutants, etc.) or may comprise anantigen-binding portion or antibody fragment (for example, a Fab,F(ab′)₂ or scFv fragment), and may be modified to effect functionality,e.g., to eliminate residual effector functions (Glu which eliminatesresidual effector functions (Reddy et al. (2000) J. Immunol.164:1925-1933).

In one embodiment, an agent capable of specifically binding hIL-8Rαcomprises a light chain variable region (LCVR) selected from the groupconsisting of SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49,53, 57, 61, 65, 69, 73, 77, and 81, or a substantially identicalfragment or sequence modified by an amino acid substitution, deletion oraddition thereof.

In one embodiment, an agent capable of specifically binding hIL-18Rαcomprises a heavy chain variable region (HCVR) selected from the groupconsisting of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47,51, 55, 59, 63, 67, 71, 75, and 79, or a substantially identicalfragment or sequence modified by an amino acid substitution, deletion oraddition thereof.

In one embodiment, an agent capable of specifically binding hIL-18Rαcomprises a LCVR encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44,48, 52, 56, 60, 64, 68, 72, 76, and 80, or a substantially identicalfragment or sequence modified by an amino acid substitution, deletion oraddition thereof.

In one embodiment, an agent capable of specifically binding hIL-18Rαcomprises a HCVR encoded by a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42,46, 50, 54, 58, 62, 66, 70, 74, and 78, or a substantially identicalfragment or sequence modified by an amino acid substitution, deletion oraddition thereof.

In another embodiment, an hIL-18Rα-binding agent comprises SEQ ID NO:3and 5; 7 and 9; 11 and 13; 15 and 17; 19 and 21; 23 and 25; 27 and 29;31 and 33; 35 and 37; or 29 and 41. Encompassed by the invention aremodifications to remove undesirable glycosylation sites, for examplesubstitution of N92 of SEQ ID NO:5 (VH1F9) to another amino acid, suchas D or E.

In an aspect of the invention, an agent capable of specifically bindinghIL-18Rα is provided, comprising a light chain CDR3 domain selected fromthe group consisting of about amino acids 89-96 of SEQ ID NO:5; about89-97 of SEQ ID NO:9 or 37; about 96-103 of SEQ ID NO:13; about 95-103of SEQ ID NO:17, 21, 25, 29, or 41; and about 95-102 of SEQ ID NO:33, ora substantially identical sequence modified by an amino acidsubstitution, deletion or addition thereof. In a further embodiment, theagent further comprises a light chain CDR1 domain selected from thegroup consisting of amino acids about 27-32 of SEQ ID NO:5, 9, or 37;and about 27-38 of SEQ ID NO:13, 17, 21, 25, 29, 33, or 41; or asubstantially identical sequence modified by an amino acid substitution,deletion or addition thereof. In a further embodiment, the agent furthercomprises a light chain CDR2 domain selected from the group consistingof amino acids about 50-52 of SEQ ID NO:5, 9, or 37; and about 56-58 ofSEQ ID NO:13, 17, 21, 25, 29, 33, or 41; or a substantially identicalsequence modified by an amino acid substitution, deletion or additionthereof.

In an aspect of the invention, an agent capable of specifically bindinghIL-18Rα is provided, comprising a heavy chain CDR3 domain selected fromthe group consisting of amino acids about 97-110 of SEQ ID NO:3; about97-117 of SEQ ID NO:7; about 97-104 of SEQ ID NO:11, 15, 19, 23, 27, 39;about 97-107 of SEQ ID NO:31, and about 100-108 of SEQ ID NO:35, or asubstantially identical sequence modified by an amino acid substitution,deletion or addition thereof. In a further aspect, the agent furthercomprises a heavy chain CDR1 domain selected from the group consistingof amino acids about 26-33 of SEQ ID NO:3, 7, 11, 15, 19, 23, 27, 31, or39; and about 26-35 of SEQ ID NO:35; or a substantially identicalsequence modified by an amino acid substitution, deletion or additionthereof. In a further embodiment, the agent further comprises a heavychain CDR2 domain selected from the group consisting of amino acidsabout 51-58 of SEQ ID NO:3, 7, 11, 15, 19, 23, 27, 31, or 39; and about53-61 of SEQ ID NO:35, or a substantially identical sequence modified byan amino acid substitution, deletion or addition thereof.

In an aspect of the invention, an agent capable of specifically bindinghIL-18Rα is provided, comprising a light chain CDR3 domain selected fromthe group consisting of amino acids about 89-96 of SEQ ID NO:5; about89-97 of SEQ ID NO:9 or 37; about 96-103 of SEQ ID NO:13, about 95-103of SEQ ID NO:17, 21, 25, 29, or 41; and about 95-102 of SEQ ID NO:33;and a heavy chain CDR3 domain selected from the group consisting ofamino acids about 97-110 of SEQ ID NO:3; about 97-117 of SEQ ID NO:7;about 97-104 of SEQ ID NO:11, 15, 19, 23, 27, or 39, about 97-107 of SEQID NO:31; and about 100-108 of SEQ ID NO:35; or a substantiallyidentical sequence modified by an amino acid substitution, deletion oraddition thereof.

In an aspect of the invention, an agent capable of specifically bindinghIL-18Rα is provided, comprising a light chain CDR1 domain selected fromthe group consisting of amino acids about 27-32 of SEQ ID NO:5, 9, or37; and about 27-38 of SEQ ID NO:13, 17, 21, 25, 29, 33, or 41; a lightchain CDR2 domain selected from the group consisting of amino acidsabout 50-52 of SEQ ID NO:5, 9, or 37; and about 56-58 of SEQ ID NO:13,17, 21, 25, 29, 33, or 41; a light chain CDR3 domain selected from thegroup consisting of amino acids about 89-96 of SEQ ID NO:5; about 89-97of SEQ ID NO:9 or 37; about 96-103 of SEQ ID NO:13; about 95-103 of SEQID NO:17, 21, 25, 29, or 41; and about 95-102 of SEQ ID NO:33; a heavychain CDR1 domain selected from the group consisting of amino acidsabout 26-33 of SEQ ID NO:3, 7, 11, 15, 19, 23, 27, 31, or 39; and about26-35 of SEQ ID NO:35; a heavy chain CDR2 domain selected from the groupconsisting of amino acids about 51-58 of SEQ ID NO:3, 7, 11, 15, 19, 23,27, 31, or 39; and about 53-61 of SEQ ID NO:35; and a heavy chain CDR3domain selected from the group consisting of amino acids about 97-110 ofSEQ ID NO:3; about 97-117 of SEQ ID NO:7, about 97-104 of SEQ ID NO:11,15, 19, 23, 27, or 39; about 97-107 of SEQ ID NO:31; and about 100-108of SEQ ID NO:35, or a substantially identical sequence modified by anamino acid substitution, deletion or addition thereof.

In an aspect, the invention features an isolated binding agent thatspecifically binds hIL-18Rα, comprising at least one complementarydetermining region (CDR), wherein the CDR is selected from the groupconsisting of (i) an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰ (SEQ ID NO:82), wherein X¹=Gly or Tyr;X²=Thr, Tyr or Asp, X³=Phe, Thr, Asn or Ser, X⁴=Thr, Phe or Val, X⁵=Ser,Asn, Thr or Ile, and X⁶=Tyr, Ser, Asn, Gly or Thr; X⁷=Tyr, Phe orabsent; X⁸=Gly, Tyr, Ser or absent; X⁹=Ala or absent; and X¹⁰=Ala orabsent; (ii) an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:83) wherein X¹=Ile, Thr or Met,X²=Asn, Arg, Ser, Thr or Tyr, X³=Pro, Thr, Ala, Val or Tyr, X⁴=Asn, Tyror Arg, X⁵=Ser or Asn, X⁶=Gly, X⁷=Asn, Gly or Trp, X⁸=Thr or Tyr, andX⁹=Asn or absent; (iii) an amino acid sequence of the formulaX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵-X¹⁶-X¹⁷-X¹⁸-X¹⁹-X²⁰-X²¹(SEQ NO:84) wherein X¹=Thr, Ala or Val, X²=Thr or Arg, X³=Pro, Asp, Glyor Glu, X⁴=Gly, Glu, Trp or Met, X⁵=Asp, Ala, Phe, Tyr, Thr or Gly,X⁶=Lys, Arg, Phe, Tyr, Asp or Ala, X⁷=Trp, Ile, Asp, Ala, Phe or Leu,X⁸=Asn, Val, Ile, Leu, Asp or Phe; X⁹=Tyr, Val, Leu, Phe, Ile or absent,X¹⁰=Tyr, Gly or absent, X¹¹=Tyr, Gly or absent, X¹²=Phe, Thr or absent,X¹³=Gln, Thr or absent, X¹⁴=Phe, Pro or absent, X¹⁵=Tyr, Trp or absent,X¹⁶=Gly, Tyr or absent, X¹⁷=Tyr or absent, X¹⁸=Gly or absent, X¹⁹=Met orabsent, X²⁰. Asp or absent, X²¹=Val or absent; (iv) an amino acidsequence of the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ IDNO:85) wherein X¹=Gln, X²=His, Asp, Ser or Thr, X³=Ile or Val, X⁴=Ser,Arg, Leu or Phe, X⁵=Ile, Asn, Tyr or Ser, X⁶=Trp, Asp, Ser or Tyr,X⁷=Ser or absent, X⁸=Asn or absent, X⁹=Asn or absent, X¹⁰=Lys or absent;X¹¹=Asn, Thr or absent, and X¹²=Tyr or absent; (v) an amino acidsequence of the formula X¹-X²-X³ (SEQ ID NO:86), wherein X¹=Ser, Ala,Trp or Asp, X² Ala or Ser, and X³=Ser; and (vi) an amino acid sequenceof the formula X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹ (SEQ ID NO:87) wherein X¹=Glnor Leu, X²=Gln or Tyr, X³=Ala, Asp, Tyr, Phe or Arg=, X⁴-Asn, Tyr, Arg,Phe or Gly, X⁵=Ser, Asn, Thr, Arg or Ile, X⁶=Phe, Tyr, Pro, Thr or Trp,X⁷=Pro, Tyr, or Trp, X⁸=Ser, Arg, Thr, Tyr or Pro, and X⁹=Thr, Ser orabsent.

In an aspect, the invention features an agent capable of bindinghIL-18Rα with an IC₅₀ of less than about 10 nM, or 500 pM, or 100 pM,preferably less than about 50 pM, more preferably less than about 20 pM,even more preferably less than about 10 pM or less than about 5 pM, asmeasured by ELISA solution affinity assay.

In an aspect, the invention provides an agent that binds hIL-18Rα with aK_(D) of less than about 20 nM, about 10 nM, about 5 nM, as measured bysurface plasmon resonance (BIAcore™) with monomeric hIL-18Rα, or about500 pM, 300 pM, 20 pM, 10 pM, or less than about 5 pM, as determined bysurface plasmon resonance with dimeric hIL-18α. In a preferredembodiment, the agent binds hIL-18Rα with a K_(D) of 20 nM or less, asmeasured by surface plasmon resonance with monomeric hIL-18Rα andexhibiting reduced cellular cytotoxicity, comprising a heavy chainselected from SEQ ID NO: 88, a SEQ ID NO: 88 variant with S229P, SEQ IDNO: 89, a SEQ ID NO: 89 variant with S223P, SEQ ID NO: 90, a SEQ IDNO:90 variant with S223P, SEQ ID NO: 91, and a SEQ ID NO: 91 variantwith S236P. In a specific embodiment, SEQ ID NO: 88-91 or a variantthereof, further comprise a light chain comprising SEQ ID NO: 4, 12, 16or 8, respectively.

In an aspect, the invention features an agent which specifically bindswithin domain 3 of hIL-18Rα, e.g., from about amino acid 220 to aboutamino acid 312 of SEQ ID NO:1. The agent of the invention may furthercomprise a label, drug or toxin, or be modified with the addition ofmolecules such as chains of polyethylene glycol.

In an aspect, the invention provides nucleic acid molecules encoding anagent of the invention. Recombinant expression vectors carrying theagent encoding nucleic acids of the invention, and host cells into whichsuch vectors have been introduced, are also encompassed by theinvention, as are methods of making the agents of the invention byculturing the host cells of the invention.

In an aspect of the invention, an agent capable of specifically bindinghIL-18Rα is provided, comprising a heavy chain CDR3 domain encoded by anucleic acid sequence selected from the group consisting of nucleotidesfrom about 289 to about 329 of SEQ ID NO:2, about 289 to about 351 ofSEQ ID NO:6; about 289 to about 312 of SEQ ID NO:10, 14, 20, 26 and 38;about 289 to about 310 of SEQ ID NO:18, about 289 to 321 of SEQ IDNO:30; and about 298 to about 324 of SEQ ID NO:34, or a sequencemodified by a nucleotide substitution, deletion or addition thereof. Ina further aspect, the agent further comprises a heavy chain CDR1 domainencoded by a nucleic acid sequence selected from the group consisting ofnucleotides from about 76 to about 99 of SEQ ID NO:2, 6, 10, 14, 22, 26,30 or 38; about 76 to about 98 of SEQ ID NO:18; and about 76 to about102 of SEQ ID NO:34, or a sequence modified by a nucleotidesubstitution, deletion or addition thereof. In a further embodiment, theagent further comprises a heavy chain CDR2 domain encoded by a nucleicacid sequence selected from the group consisting of nucleotides fromabout 151 to about 174 of SEQ ID NO:2, 6, 10, 14, 18, 20, 26, 30 or 38;and about 157 to about 183 of SEQ ID NO:34, or a sequence modified by anucleotide substitution, deletion or addition thereof.

In an aspect of the invention, an agent capable of specifically bindinghIL-18Rα is provided, comprising a light chain CDR3 domain encoded by anucleic acid sequence selected from the group consisting of nucleotidesfrom about 265 to about 288 of SEQ ID NO:4, about 265 to about 291 ofSEQ ID NO:8 and 36; about 283 to about 309 of SEQ ID NO:12, 16, 20, 24,28 or 40; about 283 to about 306 of SEQ ID NO:32, or a sequence modifiedby a nucleotide substitution, deletion or addition thereof. In a furtheraspect, the agent further comprises a light chain CDR1 domain encoded bya nucleic acid sequence selected from the group consisting ofnucleotides from about 76 to about 96 of SEQ ID NO:4, 8 and 36; about 79to about 114 of SEQ ID NO:12, 16, 24, 28, 32 or 40; and about 25 toabout 60 of SEQ ID NO:20, or a sequence modified by a nucleotidesubstitution, deletion or addition thereof. In a further embodiment, theagent further comprises a light chain CDR2 domain encoded by a nucleicacid sequence selected from the group consisting of nucleotides fromabout 148 to about 156 of SEQ ID NO:4, 8 or 36; and about 166 to about174 of SEQ ID NO:12, 16, 20, 24, 28, 32 or 40, or a sequence modified bya nucleic acid substitution, deletion or addition thereof.

In one aspect, the invention features a transgenic mouse comprising ahuman sequence encoding a LCVR or HCVR selected from the groupconsisting of SEQ ID NO: 5, 9, 13, 17, 21, 25, 29, 33, 37, or 41, andSEQ ID NO:3, 7, 11, 15, 19, 23, 27, 31, 35 or 39. In another aspect, thetransgenic mouse of the invention comprises a sequence encoding a lightchain CDR3 domain selected from the group consisting of amino acidsabout 89-96 of SEQ ID NO:5; about 89-97 of SEQ ID NO:9 or 37; about96-103 of SEQ ID NO:13, about 95-103 of SEQ ID NO:17, 21, 25, 29, or 41;and about 95-102 of SEQ ID NO:33; and a heavy chain CDR3 domain selectedfrom the group consisting of amino acids about 97-110 of SEQ ID NO:3;about 97-117 of SEQ ID NO:7; about 97-104 of SEQ ID NO:11, 15, 19, 23,27, or 39, about 97-107 of SEQ ID NO:31; and about 100-108 of SEQ IDNO:35.

In one aspect, the invention features a pharmaceutical compositioncomprising an agent which specifically binds hIL-18Rα and an acceptablecarrier.

In one aspect, the invention features methods for inhibiting hIL-18activity comprising administering a therapeutically effective amount ofan agent capable of specifically binding hIL-18Rα. In one embodiment,the method comprises contacting hIL-18Rα with an agent of the inventionsuch that hIL-18 activity is inhibited. In another embodiment, themethod comprises administering an agent of the invention to a humansubject suffering from a disorder in which IL-18 activity is detrimentalsuch that IL-18 activity in the human subject is inhibited. The disordercan be, for example, sepsis, Systemic Inflammatory Response Syndrome(SIRS), including severe sepsis, septic shock, and sepsis related tocardiac dysfunction; liver injury; arthritis, including rheumatoidarthritis; inflammatory bowel diseases, including Crohn's disease andulcerative colitis; central nervous system injury, including traumatichead injury; heart disease; hypersensitivity disorders, includingdelayed-type hypersensitivity; tumor metastasis; atherosclerosis; andperipheral vascular diseases.

Other objects and advantages will become apparent from a review of theensuing detailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

The term “human IL18Rα” (hIL-18Rα) (SEQ ID NO:1), refers to a humancytokine receptor that exists as a 60 kD membrane-associated form, thebiologically active form of which is composed of IL-18 and a dimer ofnoncovalently bound IL-18Ra and IL-18Racp. The structure of hIL-18Rα isdescribed further in, for example, Azam et al. (2003) J Immunol.171:6574-80. The term hIL-18Rα is intended to include an extracellulardomain of hIL-18Rα, which can be prepared by standard recombinantexpression methods or purchased commercially.

The agents of the invention are preferably an antibody or antibodyfragment. An “antibody” is intended to refer to immunoglobulin moleculesconsisting of four polypeptide chains, two heavy (H) chains and twolight (L) chains inter-connected by disulfide bonds. Each heavy chainhas a heavy chain variable region (HCVR or VH) and a heavy chainconstant region. The heavy chain constant region contains three domains,CH1, CH2 and CH3. Each light chain has of a light chain variable regionand a light chain constant region. The light chain constant regionconsists of one domain (CL). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “high affinity” antibody refers to those antibodies having abinding affinity to hIL-18Rα of at least 10⁻¹⁰ M; preferably 10⁻¹¹ M;even more preferably 10⁻¹² M, as measured by surface plasmon resonance(e.g., BIAcore™) or ELISA.

The term “antigen-binding portion” of an antibody (or “antibodyfragment”), refers to one or more fragments of an antibody that retainthe ability to specifically bind to an antigen (e.g., hIL-18Rα).Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VI, and VH domains of a single arm of an antibody, (v) a dAbfragment (Ward et al. (1989) Nature 241:544-546), which consists of a VHdomain, (vi) an isolated CDR, and (vii) an scFv, the two domains of theFv fragment, VL and VH, joined by a synthetic linker to form a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules. Other forms of single chain antibodies, such as diabodies arealso encompassed (see e.g., Holliger et al. (1993) PNAS USA90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibodies andHybridomas 6:93-101) and use of a cysteine residue, a marker peptide anda C-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058).Antibody portions, such as Fab and F(ab′)₂ fragments, can be preparedfrom whole antibodies using conventional techniques, such as papain orpepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hIL-18 activity”), is intended to refer to an antibody whosebinding to hIL-18Rα results in inhibition of the biological activity ofhIL-18. This inhibition of the biological activity of hIL-18 can beassessed by measuring one or more indicators of hIL-18 biologicalactivity, such as hIL-18-induced interferon gamma gene expression(either in vitro or in vivo), hIL-18-induced cellular activation andhIL-18 binding to hIL-18Rα. These indicators of hIL-18 biologicalactivity can be assessed by one or more of several standard in vitro orin vivo assays known in the art (see examples below). Preferably, theability of an antibody to neutralize hIL-18 activity is assessed byinhibition of hIL-18-induced interferon gamma production by KG-1 cellsor peripheral blood lymphocytes. As an additional or alternativeparameter of hIL-18 activity, the ability of an antibody to inhibithIL-18-induced expression of a luciferase gene fused to NFKB promoterelements, as a measure of hIL-18-induced cellular activation, can beassessed.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

The term “isolated nucleic acid molecule”, as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3) that bind hIL-18Rα is intended to refer to a nucleic acid moleculein which the nucleotide sequences encoding the antibody or antibodyportion are free of other nucleotide sequences encoding antibodies orantibody portions that bind antigens other than hIL-18Rα, which othersequences may naturally flank the nucleic acid in human genomic DNA.Thus, for example, an isolated nucleic acid of the invention encoding aVH region of an anti-hIL-18Rα antibody contains no other sequencesencoding other VH regions that bind antigens other than hIL-18Rα.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugars, carbohydrates, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous” or “substantially purified” when at least about 60 to 75%of the total protein exhibits a single species of polypeptide. Thepolypeptide or protein may be monomeric or multimeric. A substantiallypure polypeptide or protein will typically comprise about 50%, 60, 70%,80% or 90% w/w of a protein sample, more usually about 95%, andpreferably will be over 99%. Protein purity or homogeneity may bedetermined by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualizing a single polypeptide band upon staining the gel with a stainwell known in the art. For certain purposes, higher resolution may beprovided by using HPLC or other means well known in the art forpurification.

The term “polypeptide analog or variant” as used herein refers to apolypeptide that comprises a segment of at least 25 amino acids that hassubstantial identity to a portion of an amino acid sequence andspecifically binds to hIL-18Rα under suitable binding conditions and/orthe ability of hIL-18 to bind hIL-18Rα. Typically, polypeptide analogsor variants comprise a conservative amino acid substitution (orinsertion or deletion) with respect to the naturally-occurring sequence.Analogs typically are at least 20 amino acids long, preferably at least50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmutations of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence).

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (see, for example, Fauchere (1986) J. Adv. Drug Res.15:29). Systematic substitution of one or more amino acids of aconsensus sequence with a D-amino acid of the same type (e.g., D-lysinein place of L-lysine) may also be used to generate more stable peptides.In addition, constrained peptides comprising a consensus sequence or asubstantially identical consensus sequence variation may be generated bymethods known in the art (Rizo et al (1992) Ann. Rev. Biochem. 61:387,incorporated herein by reference), for example, by adding internalcysteine residues capable of forming intramolecular disulfide bridgeswhich cyclize the peptide.

The term “percent sequence identity” in the context of nucleic acidsequences refers to the residues in two sequences which are the samewhen aligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides ormore, usually at least about 18 nucleotides, more usually at least about24 nucleotides, typically at least about 28 nucleotides, more typicallyat least about 32 nucleotides, and preferably at least about 36, 48 ormore nucleotides. There are a number of different algorithms known inthe art which can be used to measure nucleotide sequence identity. Forinstance, polynucleotide sequences can be compared using FASTA, Gap orBestfit, which are programs in Wisconsin Package Version 10.0, GeneticsComputer Group (GCG), Madison, Wis. FASTA, which includes, e.g., theprograms FASTA2 and FASTA3, provides alignments and percent sequenceidentity of the regions of the best overlap between the query and searchsequences (Pearson (1990) Methods Enzymol. 183:63-98 and (2000) MethodsMol. Biol. 132:185-219, each herein incorporated by reference). Unlessotherwise specified, default parameters for a particular program oralgorithm are used. For instance, percent sequence identity betweennucleic acid sequences can be determined using FASTA with its defaultparameters (a word size of 6 and the NOPAM factor for the scoringmatrix) or using Gap with its default parameters as provided in GCGVersion 6.1, herein incorporated by reference.

The term “substantial similarity” or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, indicates that,when optimally aligned with appropriate nucleotide insertions ordeletions with another nucleic acid (or its complementary strand), thereis nucleotide sequence identity in at least about 90%, preferably atleast about 95%, and more preferably at least about 96%, 97%, 98% or 99%of the nucleotide bases, as measured by any well-known algorithm ofsequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identical” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80% sequenceidentity, preferably at least 90% or 95% sequence identity, even morepreferably at least 98% or 99% sequence identity. Preferably, residuepositions which are not identical differ by conservative amino acidsubstitutions.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “GAP” and “BESTFIT” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutant thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST. See,e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403).

Preparation of Human Antibodies

Methods for generating human antibodies include, for example,VelocImmune® (Regeneron Pharmaceuticals), XenoMouse™ technology(Abgenix), the “minilocus” approach, and phage display. The VelocImmune®technology (U.S. Pat. No. 6,596,541) encompasses a method of generatinga high specificity fully human antibody to a select antigen. Thistechnology involves generation of a transgenic mouse having a genomecomprising human heavy and light chain variable regions operably linkedto endogenous mouse constant region loci such that the mouse produces anantibody comprising a human variable region and a mouse constant regionin response to antigenic stimulation. The DNA encoding the variableregions of the heavy and light chains of the antibody are isolated andoperably linked to DNA encoding the human heavy and light chain constantregions. The DNA is then expressed in a cell capable of expressing thefully human antibody. In specific embodiment, the cell is a CHO cell.

The XenoMouse™ technology (Green et al. (1994) Nature Genetics 7:13-21)generates a mouse having both human variable and constant regions fromthe heavy chain and kappa light chain loci. In an alternative approach,others have utilized a “minilocus” approach in which an exogenous Iglocus is mimicked through inclusion of individual genes from the Iglocus (see, for example, U.S. Pat. No. 5,545,807). The DNA encoding thevariable regions can be isolated with or without being operably linkedto the DNA encoding the human heavy and light chain constant region.Other methods of generating human antibodies, including isolation from ahuman donor, are known. See, for example, U.S. Pat. No. 6,787,637(herein specifically incorporated by reference in its entirety).

Antibodies may be therapeutically useful in blocking a ligand-receptorinteraction or inhibiting receptor component interaction, rather than bykilling cells through fixation of complement and participation incomplement dependent cytotoxicity (CDC). The constant region of anantibody is important in the ability of an antibody to fix complementand participate in CDC. Thus, the isotype of an antibody may be selectedon the basis of whether it is desirable for the antibody to fixcomplement. Generally, in the present case, it is not required for theantibodies of the invention to fix complement and participate in CDC,and thus desirable isotypes include human IgG2 and IgG4.

Human immunoglobulins can exist in two forms that are associated withhinge heterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via interchain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a single lightand heavy chain. These forms have been extremely difficult to separate,even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. In fact, asingle amino acid substitution in the hinge region of the human IgG4hinge can significantly reduce the appearance of the second form (Angalet al. 1993. Molecular Immunology 30: 105) to levels typically observedusing a human IgG1 hinge. The instant invention encompasses antibodieshaving one or more mutations in the hinge, CH2 or CH3 region, which maybe desirable, for example, in production to improve the yield of thedesired antibody form.

Antibodies of the invention are prepared with the use of VelocImmune®technology. A transgenic mouse in which the endogenous immunoglobulinheavy and light chain variable regions are replaced with thecorresponding human variable regions is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) recovered from the micethat express antibodies. The lymphatic cells may be fused with amyeloid-type cell line to prepare immortal hybridoma cell lines, andsuch hybridoma cell lines are screened and selected to identifyhybridoma cell lines that produce antibodies specific to the antigen ofinterest. DNA encoding the variable regions of the heavy chain and lightchain may be isolated and linked to desirable isotypic constant regionsof the heavy chain and light chain. Such an antibody protein may beproduced in a cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies may be isolated directly fromantigen-specific lymphocytes.

In one embodiment, the transgenic mouse comprises up to 12 functionalhuman variable heavy chain genes and up to 11 functional human variablekappa light chain genes (MI). In another embodiment, the transgenicmouse comprises from 25 to 30 human variable heavy chain genes and from18 to 20 human variable kappa light chain genes (MII). In yet anotherembodiment, the transgenic mouse comprises from 43 to 48 human variableheavy chain genes and from 20 to 22 human variable kappa light chaingenes (MIII). In yet another embodiment, the transgenic mouse comprisesabout 80 human variable heavy chain genes and about 40 human variablekappa light chain genes (MVIII).

In general, the antibodies of the instant invention possess very highaffinities, typically possessing K_(D)s of from about 10⁻⁹ through about10⁻¹¹ M, when measured by binding to antigen either immobilized on solidphase or in solution phase.

Epitope Mapping and Related Technologies

To screen for antibodies which bind to a particular epitope (e.g., thosewhich block binding of IgE to its high affinity receptor), a routinecross-blocking assay such as that described in Harlow and Lane,Antibodies (supra) can be performed. Other methods include alaninescanning mutants, peptide blots (Reineke (2004) Methods Mol Biol248:443-63) (herein specifically incorporated by reference in itsentirety), or peptide cleavage analysis. In addition, methods such asepitope excision, epitope extraction and chemical modification ofantigens can be employed (Tomer (2000) Protein Science: 9: 487-496,herein specifically incorporated by reference in its entirety).

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Antibodiesthat recognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen.

Functional Analysis

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (USpatent application No. 2004/0101920, herein specifically incorporated byreference in its entirety). Each category may reflect a unique epitopeeither distinctly different from or partially overlapping with epitoperepresented by another category. This technology allows rapid filteringof genetically identical antibodies, such that characterization can befocused on genetically distinct antibodies. When applied to hybridomascreening, MAP may facilitate identification of rare hybridoma clonesthat produce mAbs having the desired characteristics. MAP may be used tosort the hIL-18Rα antibodies of the invention into groups of antibodiesbinding different epitopes.

The antigen protein may be immobilized on either biosensor chip surfacesor polystyrene beads. The latter can be processed with, for example, anassay such as multiplex Luminex™ detection assay (Luminex Corp., Austin,Tex.). Because of the capacity of Luminex™ to handle multiplex analysiswith up to 100 different types of beads, Luminex™ provides almostunlimited antigen surfaces with various modifications, resulting inimproved resolution in antibody epitope profiling over a biosensorassay.

EXAMPLES

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Human IL-18Rα

Immunization of rodents can be done by any methods known in the art(see, for example, Harlow and Lane, Antibodies (supra); Malik andLillehoj, Antibody techniques: Academic Press, 1994, San Diego). In apreferred embodiment, antigen is administered directly to mice whichcomprise DNA loci encoding both human Ig heavy chain variable region andkappa light chain variable region (VelocImmune®), with an adjuvant tostimulate the immune response. Such an adjuvant includes complete andincomplete Freund's adjuvant, MPL+TDM adjuvant system (Sigma), or RIBI(muramyl dipeptides) (see O'Hagan (2000) Vaccine Adjuvant, Humana Press,Totawa, N.J.). Such an adjuvant can prevent rapid dispersal ofpolypeptide by sequestering the antigen in a local depot, and maycontain factors that can stimulate host immune response. In anotherpreferred embodiment, the antigen is administered indirectly as DNAplasmid that contains an hIL-18Rα gene and expresses hIL-18Rα using thehost cellular protein expression machinery to produce antigen in vivo.In both approaches, the immunization schedule requires severaladministrations spaced by a few weeks. The antibody immune response ismonitored by standard antigen specific immunoassay. When animals reachedtheir maximum immune response, the antibody expressing B cells wereharvested and fused with mouse myeloma cells to preserve theirviability, forming hybridoma cells. To select functionally desirableantibody expressing cells, conditioned medium of the cells was screenedagainst their antigen specificity as well as preferred epitope orfunctionality by hIL-18Rα binding affinity and preferred epitope ofblocking hIL-18 binding to hIL-18Rα by surface plasmon resonance(BIAcore®) and immunoassays, and in vitro IL-18 neutralization efficacy,as described below.

Antibodies were generated as described above, 10 of which are shown inamino acid sequences SEQ ID NO:5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45,49, 53, 57, 61, 65, 69, 73, 77, and 81 (LCVR) and SEQ ID NO: 3, 7, 11,15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, and 79,(HCVR).

Example 2 Affinity Binding and Blocking Determinations

The K_(D) of the antigen binding to the selected antibodies describedabove were determined by surface kinetics on a real-time biosensorsurface plasmon resonance assay (BIAcore™). The antibody derived fromhybridoma was captured on an anti-mouse IgG surface and exposed tovarious concentrations of hIL-8Rα-hFc fusion protein. Kinetic analysisusing evaluation software was performed to obtain the association anddissociation rate constants. Results are shown in Table 1.

Binding affinity of the antibodies to antigen was also measured using animmunoassay. Antibodies were characterized from hybridoma conditionedmedium or as purified protein. Protein was purified after hybridomacells were grown in low IgG medium and affinity purified using ProteinG. IC₅₀ of antigen binding was determined with an ELISA-based assay.Briefly, constant amounts of antibody at different levels, either inhybridoma conditioned medium or purified, were premixed with serialdilutions of antigen protein, hIL-18Rα-hFc, ranging from 0 to 10 mg/ml,and incubated for two hours at room temperature to reach pseudo-bindingequilibrium between the antibody and antigen. These solutions were thentransferred to 96-well hIL-18Rα-hFc pre-coated plates (solid phase) toallow the free-antibody in the mixtures to bind to plate-coatedhIL-18Rα-hFc. The plates were typically coated with 1 to 2 μg/mlhIL-18Rα-hFc protein in PBS solution overnight at 4° C. followed by BSAnonspecific blocking. After washing off excess antibody in solution, theplate-bound antibodies were detected with an HRP-conjugated goatanti-mouse IgG polyclonal antibody reagent and developed using eithercolorimetric or chemiluminescent substrates. The dependency of thesignals on the concentrations of antigen in solution was analyzed with a4 parameter fit analysis using Prism™ software from Graph Pad™ andreported as IC₅₀ that render 50% reduction of antibody binding toplate-coated antigen. Results are showed in Table 1 (*conditionedmedium).

TABLE 1 Antigen Binding Affinity Antibody ELISA IC₅₀ nM BIAcore ™ K_(D)nM* VK8E1 0.017 0.01 VK5G1 0.002 0.052 VK2C2 0.007 0.003 VK3H5 0.0070.002 VJ7D5 0.003 0.005 VK2E5 0.010 0.001 VI2H10 1.7 0.495 VH1F9 0.0260.135 VI2C4-11 0.009 0.054 VI2C4-18 0.019 0.079 VI2A5 0.011 0.002

Conversion of hybridoma-derived chimeric IgG of human variable and mouseconstant regions in both heavy and light chains to fully human IgG didnot affect antigen binding. Both mouse IgG1 and human IgG4 versions ofthe derived antibody were measured to determine K_(D) for monomericantigen using BIAcore™ (Table 2). Binding affinity of monomeric receptorwas measured by capturing mAb from subclone hybridoma supernatant andCHO conditioned media over amine coupled anti-mouse IgG and anti-humanIgG. Varying concentrations of 50 nM-12.5 nM of monomeric receptor,hIL18Ra-hIL-18Rb-myc-myc-his, were injected over the captured surface.Data were processed and analyzed using a 1:1 binding model and K_(D)values were calculated.

TABLE 2 Chimeric Mouse IgG and Fully Human IgG Binding to MonomericAntigen Ab Isotype K_(D) (nM) VH1F9-18 human IgG4 6.00 VH1F9-18 mouseIgG1 6.81 VK5G1-15 human IgG4 4.80 VK5G1-15 mouse IgG1 4.68 VK-2C2-22human IgG4 2.16 VK-2C2-22 mouse IgG1 2.18 VI-2H10-7 human IgG4 19.20VI-2H10-7 mouse IgG1 17.00

The ability of the test antibody to block (inhibit) hIL-18 binding tothe IL-18Rα receptor was determined using surface plasmon resonance.Purified antigen IL-18R1-hFc molecules were captured by goat anti-humanIgG polyclonal antibodies that were immobilized on a CM-5 surfacethrough amine coupling chemistry, to a density of 250 RU. An IL-18solution, 0.25 ml at 50 nM, was then injected over the receptor surfaceand the bound IL-18 was recorded (first injection of hIL-18). The boundhIL-18 was then removed with a pulse of 3 M MgCl₂ following byconditioning buffer. Anti-hIL18R1 antibody in hybridoma-conditionedmedium was then injected over the captured receptor surface followed bya second injection of hIL-18. The percent reduction in hIL-18 bindingresulting from preformed antibody and receptor complex was used as ascore to define IL-18 blockers (antagonists) from non-blockers. Resultsare shown in Table 3 (*conditioned medium).

TABLE 3 Identification of IL-18 Blockers Antibody % Blocking* VK8E1 96VK5G1 92 VK2C2 98 VK3H5 96 VJ7D5 91 VK2E5 98 VI2H10 82 VH1F9 96 VI2C4-1141 VI2C4-18 41 VI2A5 0

Example 3 In Vitro Neutralization of hIL-18 Activity

An HEK293/NFkB-luciferase bioassay was used to determine the ability ofthe hIL-18Rα-specific antibodies of the invention to block the activityof hIL-18. Human embryonic kidney 293 cells (HEK293) were transfectedwith an NFκB-luciferase reporter plasmid. By placing an NFκB promoterelement upstream of the luciferase gene one can monitor NFκB activity incells. When cells containing the HEK293/NFκB luciferase construct andthe IL-18 receptors were stimulated with hIL-18, the luciferase gene wasexpressed and luciferase activity detected in cell lysates. A stabletransfected cell line, HEK293/D9, was first selected for good responseto IL-1β as detected by luciferase activity and then transfected withthe hIL-18Rα (IL-IRrp) and IL-18Rβ (IL1RAcPL) receptors to producehIL-18-NFκB-luciferase cells.

For the assay, hIL-18-NFκB-luciferase cells were suspended at 1.25×10⁵cells per ml in medium and 0.08 ml of cells plated at 10,000 cells perwell in a 96-well tissue culture plate. Plates were incubated for ˜16hours at 37° C. in a humidified 5% CO₂ incubator. Hybridoma supernatantswere quantified by an antigen-specific ELISA. 0.05 ml of serialdilutions, 0-10 nM, of the hIL-18Ralpha antibodies were added to the96-well plate containing the hIL-18-NFκB-luciferase cells. After a 1hour incubation, 2 pM of recombinant human IL-18 was added. Controlwells contained no hIL-18Rα-specific antibodies. After incubation at 37°C. for 6 hours in a humidified 5% CO₂ incubator, the plates wereequilibrated to room temperature for ˜30 minutes and 130 μl ofSteady-Glo™ luciferase substrate (Promega) was added. Luminescenceintensity was measured using a multilabel counter, IC₅₀, which is a 50%reduction in hIL-18 stimulated activity, were determined with a 4parameter fit analysis using Prism™ software from Graph Pad™. Table 4shows the bioassay IC50 values of the IL-18Rα hybridoma supernatants(Prot G purified protein).

KG-1 bioassay. The KG-1 bioassay is an endogenous bioassay used todetermine blocking of IL-18 signaling. KG-1 cells are a myelogenousleukemia-derived cell line with endogenous IL18Rα and β receptors thatwhen stimulated with the addition of IL-18, produce IFNγ. For the assayKG-1 cells were washed twice with media and suspended at 6×10⁵ cells perml in medium and 30,000 cells per well were plated in a 96-well tissueculture plate. Plates were incubated for ˜24 hours at 37° C. in ahumidified 5% CO₂ incubator. rhTNFa was added to 40 ng/ml as aco-stimulator along with 0.05 ml of serial dilutions, 0-12.5 nM, of thehIL-18Rα-specific antibodies and plates were incubated for 1 hour 37° C.in a humidified 5% CO₂ incubator. 0.025 ml of hIL-18 was added to thewells at a final concentration of 90 pM and the well volume was adjustedto 0.2 ml with media. Control wells contained no hIL-18Rα-specificantibody. Cells were incubated for 24 hours 37° C. in a humidified 5%CO₂ incubator. Media from the wells was analyzed to determine the amountof interferon gamma production using an OptE1A ELISA (BD cat #555142).

Standard curves were generated by linear regression of standards usingGraphPad Prism™ to determine X from Y to get pg/ml values for cellsupernatants by OD. Values of interferon gamma were analyzed vs Log Mconcentrations of antibody by non-linear regression analysis sigmoidaldose response to give an IC50 value for antibody inhibition of hIL-18bioactivity. Table 4 shows the bioassay IC₅₀ values of the IL-18Rαhybridoma supernatants (Prot G purified protein).

TABLE 4 In Vitro Luciferase and KG-1 Bioassays Antibody LuciferaseBioassay (IC50 nM) KG-1 Bioassay (IC50 nM) VK8E1 0.331 0.025 VK5G1 0.1660.028 VK2C2 0.139 0.031 VK3H5 0.134 0.040 VJ7D5 0.194 0.064 VK2E5 0.3360.047 VI2H10 0.196 0.064 VH1F9 0.653 0.085 VI2C4-11 1.06 0.125 VI2C4-180.722 0.132 VI2A5 2.58 0.21

Example 4 Epitope Mapping Analysis

Preliminary studies using monoclonal antibodies raised against hIL-18Rα(Swiss-Prot entry Q13478) have shown that these antibodies inhibit thebinding of hIL-18 to the receptor. Use of a variety of in vitro proteasedigestions of the recombinant hIL-18 receptor ecto-domain fused tomurine Fc has revealed the binding epitope of these monoclonals to becontained near or within the third immunoglobulin domain (residues220-312 as defined by Swiss-Prot) of the hIL-18 receptor extracellulardomain.

Using Ficin protease digestion, 4 Coomassie stained bands wereidentified for both non-reduced (200, 140, 55 and 42 kD) digests andreduced (95, 60, 42 and 30 kD) digests. Comparing the data obtained fromanalysis of the 60 kD vs 42 kD reduced bands using a combination ofimmunodetection and N-terminal protein sequencing, a 60 kD band wasobserved which possessed immunoreactivity towards the anti-IL18R1antibodies, while the 42 kD band revealed loss of anti-IL18 receptorecto-domain immunoreactivity. Since the observed peptide sequence ofboth 60 kD and 42 kD bands were identical and started with the firstamino acid of the mature protein (residue #19 by Swiss-Prot), thisimplies that a ˜18 kD fragment of the C-terminal portion of theecto-domain was cleaved off to yield the 42 kD band and that this ˜18 kDfragment presumably contains the antibody epitope and would thereforeexplain the loss of immunoreaction with the antibodies.

A similar strategy was implemented using two other proteases, namelytrypsin and endo-GluC. Digestion of the receptor using trypsin yieldedmajor fragments of 26, 19, and 15 kD (non-reduced), and 30 and 26 kD(reduced). All but the 15 kD fragment, which was sequenced and shown toarise from Ig-domain 1, showed immunoreactivity towards the antibodies.The 4 remaining bands (26, 19 and 26, 30 kD) showed not onlyimmunoreactivity but also had the same peptide sequence, starting withS-N-I-V-etc. This sequence begins at residue 211 (Swiss-Prot sequence),near the predicted start of Ig-domain 3.

Using an endo-GluC digest, a dominant Coomassie stained band wasobserved at approximately 40 kD on a reduced gel. This band was reactivewith antibodies to murine-Fc, although no reactivity was observed withanti-ILL 8 receptor antibodies. The N-terminal peptide sequence of thisfragment, G-K-W-H-etc., begins at residue 274 (Swiss-Prot sequence)which is on the carboxy-terminal half of Ig-domain 3. This observationwould imply that the epitope region does not extend C-terminal toresidue 274.

1. An isolated antibody or antibody fragment capable of specificallybinding human interleukin-18 receptor alpha (hIL-18Rα), wherein theantibody comprises a light chain variable region (LCVR) comprising SEQID:
 17. 2. The antibody or antibody fragment of claim 1, characterizedby binding hIL-18Rα with an IC50 of about 2 pM, as measured by ELISAsolution affinity assay.
 3. The antibody or antibody fragment of claim1, characterized by binding hIL-18Rα with a KD of less than 300 pM, asdetermined by surface plasmon resonance.
 4. The antibody or antibodyfragment of claim 3, characterized by binding hIL-18Rα with a KD ofabout 52 pM, as determined by surface plasmon resonance.
 5. The antibodyor antibody fragment of claim 1, further comprising a heavy chainvariable region (HCVR) comprising SEQ ID NO:
 15. 6. A pharmaceuticalcomposition comprising the antibody or antibody fragment of claim 1 anda pharmaceutically acceptable carrier.
 7. An isolated antibody orantibody fragment capable of specifically binding human interleukin-18receptor alpha (hIL-18Rα), wherein the antibody comprises a HCVRcomprising SEQ ID NO:
 15. 8. The antibody or antibody fragment of claim7, characterized by binding hIL-18Rα with an IC50 of about 2 pM, asmeasured by ELISA solution affinity assay.
 9. The antibody or antibodyfragment of claim 7, characterized by binding hIL-18Rα with a KD of lessthan 300 pM, as determined by surface plasmon resonance.
 10. Theantibody or antibody fragment of claim 9, characterized by bindinghIL-18Rα with a KD of about 52 pM, as determined by surface plasmonresonance.
 11. A pharmaceutical composition comprising the antibody orantibody fragment of claim 7 and a pharmaceutically acceptable carrier.12. An isolated antibody or antibody fragment capable of specificallybinding human interleukin-18 receptor alpha (hIL-18Rα), wherein theantibody comprises a LCVR comprising the three CDRs from SEQ ID NO:17,and a HCVR comprising the three CDRs from SEQ ID NO:15.
 13. The antibodyor antibody fragment of claim 12, wherein light chain CDR1, CDR2 andCDR3 consist of, respectively, amino acids 27-38, 56-58 and 95-103 ofSEQ ID NO:17.
 14. The antibody or antibody fragment of claim 12, whereinheavy chain CDR1, CDR2 and CDR3 consist of, respectively, amino acids26-33, 51-58 and 97-104 of SEQ ID NO:
 15. 15. A pharmaceuticalcomposition comprising the antibody or antibody fragment of claim 12 anda pharmaceutically acceptable carrier.